DNA removal

We have so far described the structure of DNA as an extended double helix The crys tallographic evidence that gave rise to this picture was obtained on a sample of DNA removed from the cell that contained it Within a cell—its native state—DNA almost always adopts some shape other than an extended chain We can understand why by doing a little arithmetic Each helix of B DNA makes a complete turn every 3 4 X 10 m and there are about 10 base parrs per turn A typical human DNA contains 10 base parrs Therefore... 

Alkaline phosphatase 1 Dephosphorylates 5 ends of RNA and DNA. Removal of S -P04 groups prior to kinase labeling to prevent self-ligation. 

SI nuclease j Degrades single-stranded DNA. Removal of "hairpin" in synthesis of cDNA RNA mapping studies (both 5 and 3 ends). 

DNA chain elongation is catalyzed by DNA polymerase III using 5 -deoxyribonucleoside triphosphates as substrates. The enzyme "proofreads" the newly synthesized DNA, removing terminal mismatched nucleotides with its 3 —>5 exonuclease activity. 

Add 10 mL hybridization buffer, (6X SSPE, 50% formamide, 0.5% SDS, 10% dextran, and 50 ug/ml. of sonicated salmon sperm or herring sperm DNA). Remove air and bubbles and seal by heating the open side. Incubate at 37°C for 1 h, open bag by cutting one comer, and drain. 

TABLE 4 Effect of Flow Rate on Efficiency of DNA Removal (Single layer)... 

MYH MutY homolog DNA Removes adenine opposite 8-oxoG, 2-OH-A opposite G... 

To terminate the assay and harvest samples for quantitative analysis of HBV virion DNA, remove the culture medium 24 h following d 9 of treatment, and store the culture medium in 96-well U-bottomed culture plates. Cover the U-bottomed plates tightly with parafilm to prevent evaporation. Store these samples at 4°C until blotting is performed. Samples can eventually be transferred to -20°C for long-term storage. 

Add 50 ng 32P-labeled DNA probe to carrier DNA and heat to 100°C for 10 min. Add to 10 mL hybridization mix (3X SSC, 2% SDS, 5X Denhardf s, 0.2 mg/mL sheared carrier DNA). Remove prehybridization solution and replace with hybridization solution containing probe. Incubate in hybridization oven or shaker overnight at 65°C. 

To determine the 5 -ends of an mRNA molecule, a 5 -labeled ss DNA probe is hybridized to the RNA in which a overhang of the 3 -end of the DNA and a hybridized 5 -end of the DNA is obtained. Digestion with SI nuclease (ss specific for both RNA and DNA) removes the 3 -overhang. The SI enzyme is eliminated and the exact size of the SI probe, protected by the mRNA, is determined on a sequencing gel using a sequence ladder as a marker. Similarly, the 3 -ends of an mRNA molecule can be established using a 3 -labeled ss DNA probe. 

The PCR (Fig. 10) allows small segments of genomic DNA removed from crude cell lysates to be amplified in quantities sufficient for subsequent analysis. This is achieved by using a succession of incubation steps at different temperatures. Fundamental to the procedure are a pair of oligonucleotides, or primers, that are made synthetically with nucleotide sequences complementary to those of the 3 and 5 ends of the gene segment of interest. One primer corresponds to the 3 end and one to the 5 end. The DNA sample is first heated to 94°C to convert it from... 

MutSot binds to G4 DNA with affinity even higher than for the best of its documented substrates in canonical mismatch repair (apparent = 1 nM). This raises the possibility that MutSa directs a pathway for G4 DNA removal. However, downstream factors in this pathway may very well be distinct from those that typically participate in mismatch repair, because binding to G4 DNA is not sensitive to addition of ATP. Interestingly, the RecQ family helicase BLM interacts with and is stimulated by MutSa. This raises the possibility that MutSa might stimulate G4 DNA unwinding, rather than excision of quadruplex structures. 

As an alternative to nuclease digestion, cationic detergents can be added during lysis to selectively precipitate cellular DNA. Recent experiments with the detergent domiphen bromide have shown that it is possible to obtain three logs of DNA clearance without losses in the infectivity of purified viral particles [107]. Thus, the use of DNA removal operations such as nuclease treatment and anion exchange chromatography may be eliminated or reduced. 

After treatment with UDGase when uracil is present in the DNA strand, there is a significant shift in retention time of the modified single-stranded DNA. Removal of the uracil base from the single-stranded DNA results in elution at an earlier retention time through loss of the uracil base and subsequent decrease in hydro-phobicity of the DNA. 

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